Hydrogelation agents

ABSTRACT

A gelatinizer is provided having a component which promises to have biological compatibility, which can easily be mass-produced by a simple method, and which can solidify a large amount of water or aqueous solution when only a very small weight of it is used. 
     This invention is a hydrogelatinizer represented by the general formula:
 
R-A n 
 
where A, which may be identical or different, are respectively nucleotides, n is 2 or 3, and R is a hydrocarbon chain (when n is 2, R is bivalent, and when n is 3, R is trivalent), said hydrocarbon being bonded to a phosphoric acid part of said nucleotides.

FIELD OF THE INVENTION

This invention relates to a hydrogelatinizer comprising a nucleotidewhich is a component monomer of DNA, which can solidify 500 times ormore its own weight of an aqueous solution using a very small amount ofa nucleotide lipid wherein this nucleotide component is connected by ahydrocarbon chain, and to a method of manufacturing thishydrogelatinizer.

DESCRIPTION OF THE RELATED ART

Hydrogels using polymer gelatinizers such as polyacrylic acid are knownin the related art. However, hydrogels produced by these polymergelatinizers are referred to as irreversible physical gels which, onceformed, do not revert to the original water. The physical properties ofthe resulting gel such as its hardness and thermal stability cannot becontrolled, and these gelatinizers furthermore have no effect on liquidscontaining hydrophilic organic solvents such as alcohol.

There are very few low molecular weight hydrogelatinizers, examplesbeing long chain dicarboxylic acids and bisurea (e.g., L. A. Estroff andA. D. Hamilton, Angew.Chem.Int.Ed., 39, 3447–3450 (2000); F. M. Mengerand K. L. Caran, J.Am.Chem.Soc.,122,11679–11691 (2000)).

However, when these related art hydrogels are used as biologicalcompatibility materials, or as gel materials for separating proteins orDNA, their reactivity and toxicity were a problem.

Problems Which this Invention Aims to Solve

This invention aims to provide a gelatinizer having a component whichpromises to have biological compatibility, which can easily bemass-produced by a simple method, and which can solidify a large amountof water or aqueous solution when only a very small weight of it isused.

Means to Solve the Above Problems

The Inventors carried out detailed studies of methods for manufacturinghydrogelatinizers comprising effective biological material components.As a result of the studies, they discovered that a hydrogel could bemanufactured having a nucleotide which is an important component monomerof genetic DNA at the end of the molecule. They further discovered that,by dissolving a nucleotide lipid wherein these (components) areconnected by hydrocarbon chains in an aqueous solution, heating, andallowing to stand, water could be solidified with an extremely smallcomponent ratio, and based on this discovery, they arrived at thepresent invention.

The present invention is therefore a hydrogelatinizer represented by thegeneral formula:R-A_(n)where A, which may be identical or different, are respectivelynucleotides, n is 2 or 3, and R is a hydrocarbon chain (when n is 2, Ris bivalent, and when n is 3, R is trivalent), said hydrocarbon beingbonded to a phosphoric acid part of said nucleotides.

Further, the invention is a hydrogelatinizer represented by the generalformula:B—R—Cwhere, B and C, which may be identical or different, are respectivelynucleotides and R is a bivalent hydrocarbon chain, said hydrocarbonbeing bonded to a phosphoric acid part of said nucleotides.

The nucleotide may be monophosphoric acid, the number of carbon atoms inR may be 12–20, and the nucleotide may be2′-deoxythymidine-3′-monophosphoric acid.

The invention is also a method of manufacturing the hydrogelatinizeraccording to any of claims 1–4, which comprises the steps of

1) reacting a nucleotide, comprising a sugar part protected by an acetylgroup, 5′-O-4,4′,4″-tris(4-benzoyloxy)trityl group or dimethoxytritylgroup, with a diol or triol to produce a phosphite ester,

2) oxidizing this phosphite ester with iodine or t-butyl hydroperoxideto produce a phosphate ester, and

3) removing the protective group by using an acid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing a ¹H-NMR spectral chart of1,12-(dodecanedioxy)bis(3′-phosphatidyl-2′-deoxythymidine) obtained inthe Manufacturing Example 1.

FIG. 2 is a diagram showing a scanning electron micrograph of afreeze-dried hydrogel of Example 3.

DETAILED DESCRIPTION OF THE INVENTION

The gelatinizer of this invention is represented by the followinggeneral formula:R-A_(n)where n is 2 or 3, but preferably 2.

A is a nucleotide of which n are present in one molecule of thegelatinizer. These nucleotides may be identical or different. Thenucleotide may be a ribonucleotide or a deoxyribonucleotide, and themolecule may contain one, two or more thereof at positions 3 or 5. Fromthe viewpoint of ease of manufacture of the gelatinizer of thisinvention, phosphoric acid is preferably bonded at position 3 of thedeoxythymidine. As the solubility in water of the gelatinizer increaseswhen these phosphoric acid units increase, although there is a balancewith the size of the hydrophobic hydrocarbon chain, the gel tends to bemore difficult to produce if the solubility of the gelatinizer in wateris too high. Therefore, the number of phosphoric acid units ispreferably small, and diphosphoric acid or more preferablymonophosphoric acid is preferred to triphosphoric acid.

R is a hydrocarbon chain. When n is 2, the hydrocarbon chain isbivalent, and when n is 3, the hydrocarbon chain is trivalent. Thishydrocarbon chain has the function of imparting hydrophobic propertiesto the gelatinizer, and due to the balance with the aforesaidhydrophilic phosphoric acid, it confers a gelatinizing ability on thegelatinizer. Therefore, there is no particular limitation on thehydrocarbon, which may be straight chain, branched or cyclic. Further,the bonding sites (or bonding hands) (when n=2, two sites, and when n=3,three sites) locate preferably at the end of the hydrocarbon chain.

The number of carbon atoms in this hydrocarbon chain is preferably12–20, but more preferably 18–20. It is preferred that this hydrocarbonchain is an oligomethylene group, and in particular —(CH₂)_(m)— (m ispreferably 12–20, more preferably 18–20) having the bonding sites at theend.

In this gelatinizing molecule, the hydrocarbon chain is bonded via thephosphoric acid part of the nucleotide. When the nucleotide containsplural phosphoric acid units, the nucleotide may be the oligomer(hydrocarbon chain-nucleotide)_(l) (where l is an integer).

The nucleotide is not necessarily of only one type, and two or moretypes may be used. Likewise, two or more types of the hydrocarbon chainmay be used.

A specific example of this gelatinizer is the gelatinizer represented bythe following formula:

(in the formula, n is 12–20). Of these, it is preferred that n is 18 or20 as a very small amount can then solidify a large amount of water interms of weight ratio.

The gelatinizer this invention may be manufactured by reacting anucleotide, having a sugar part protected, with a diol or triol toproduce a phosphite ester, oxidizing this phosphite ester with iodine ort-butyl hydroperoxide to produce a phosphate ester, and removing theprotective group by using an acid. When this nucleotide and diol ortriol are reacted, if the phosphoric acid part of the nucleotide isprotected by an amidite or the like previously, the reactivity of thephosphoric acid part increases, so the target product can easily beobtained by performing an exchange reaction between this and the diol ortriol.

For example, it may be manufactured by bonding a long chain diolrepresented by HO—(CH₂)_(n)—OH (in the formula, n is an integer in therange 12–20) with the dioxythymidine phosphoramidite represented by thefollowing formula:

to obtain a phosphite ester, which is converted to a phosphate triesterby an oxidation reaction, and finally removing the protective group.

Examples of the protective group of the sugar part of the nucleotide areacetyl, 5′-O-4,4′,4″-tri (4-benzoyloxy)trityl and dimethoxytrityl, butdimethoxytrityl can efficiently and easily be removed, and is thereforeconvenient. For the oxidation reaction, iodine or t-butyl hydroperoxidemay be used, but t-butyl hydroperoxide is preferred from the viewpointof purification. Examples of reagents used to remove the dimethoxytritylprotective group are acetic acid, phosphoric acid, hydrochloric acid,trichloroacetic acid and trifluoracetic acid, but trifluoroacetic acidis preferred from the viewpoint of yield.

Next, the method of manufacturing a hydrogel will be described. Thegelatinizer of the invention is dissolved in an aqueous solution. Aweakly acidic (pH 4) or weakly alkaline (pH 10) aqueous solution ispreferred from the viewpoint of solubility. If the pH of the aqueoussolution is less than 4, all of the phosphoric acid part is protonated,and it is difficult to dissolve in water. If the pH is higher than 10,the phosphoric acid part dissociates, and remains dissociated so thatthe water is not solidified. After dissolving the compound in theaqueous solution with heating, it is gradually cooled to roomtemperature and allowed to stand. In the dissolution process, heat aloneis sufficient if the compound can be completely dissolved. If necessary,the compound can be efficiently converted to a simple dispersion in anaqueous solvent by ultrasound. The heating time is of the order of 30minutes to 2 hours, but to ensure that dissolution of the compound iscomplete, it is preferably one hour or more. When the aqueous solutionin which the compound has been dissolved is gradually cooled in air, andleft at room temperature, the aqueous solution solidifies within oneday-several days, and forms a hydrogel. The formation of the gel can beconfirmed by inverting a test tube containing the hydrogel and observingthat the gel does not flow downwards. The fluidity of the resulting gelmay be varied by adjusting the pH of the water and the concentration ofthe gelatinizer during manufacture. In general, the formation of thehydrogel may be observed with the naked eye, but if the microstructureof the gel is observed using an optical microscope or scanning electronmicroscope, it can be seen that extremely fine fibers of the order ofnanometers are entangled together to form a lattice structure.

Even a very small amount of the hydrogelatinizer of this invention isable to solidify a large amount of water, and the softness, stabilityand water retention amount of the resulting gel can be freely varied byadjusting the manufacturing conditions when it is formed. Further, as ithas no toxicity, it is particularly suitable for use as a biologicalcompatibility material, structural or culture matrix material, or as agel for separating biological materials such as proteins and nucleicacids. As in the case of ordinary hydrogels, it may also be used as awater retaining agent (desert greenification or cultivating plants), ora moisture absorbent (pet tray urine absorbent, physiological waterabsorbent). In addition, it has application as a moisturizing agent inthe fine chemical industry, pharmaceuticals and cosmetics, and has greatindustrial value.

EXAMPLES

This invention will now be described referring to specific examples, butit should be understood that the invention is not be construed as beinglimited in any way thereby. The Rf value in thin layer chromatographywas Rf1 when a chloroform/methanol (volume ratio 4/1) mixed solvent wasused as developing solvent.

Manufacturing Example 1

2.3 g (9.4 mmol) of 1,10 decane dicarboxylic acid, 3.3 ml (28 mmol) ofthionyl chloride and one drop of N,N-dimethylformamide were added todichloroethane, and the mixture was heated under reflux for two hours.After the reaction, the acid chloride obtained by completely distillingoff the solvent under reduced pressure was dissolved in tetrahydrofuran.Next, 0.5 g (13 mmol) of lithium aluminium hydride was added to thetetrahydrofuran, and the mixture kept at −50° C. After graduallydripping the aforesaid acid chloride solution into this solution, themixture was returned to room temperature, and heated under reflux for 3hours. Subsequently, it was stirred at room temperature for 24 hours,and ethyl acetate followed by a saturated aqueous solution of sodiumsulphate was added until the bubbles disappeared. Next, the reactionsolution was placed under reduced pressure to distill off the solvent,the solid obtained was suspended in chloroform and filtered, and thefiltrate was distilled under reduced pressure. The solid obtained wasrecrystallized from a solution of hexane/ethyl acetate=2/1, and 1.5 g of1,12-dodecane diol was obtained as a white solid (yield=74%).

0.14 g (0.7 mmol) of this 1,12 dodecane diol and 0.2 g (3 mmol) of1H-tetrazole were dissolved in tetrahydrofuran, and 1.0 g (1.4 mmol) of5′-O-dimethoxytrityl-2′-deoxythymidine-3′-O——[O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite]was added. After stirring at room temperature for 24 hours, 0.4 ml of a70% aqueous solution of t-hydroperoxide was added to the reactionsolution, and stirred for one hour. Next, 8 ml of a 28% aqueous solutionof ammonia was added and stirred for 24 hours, the solvent was distilledoff under reduced pressure, and the solid obtained was purified bysilica gel column chromatography (eluent: chloroform/methanol=4/1). Thesolid obtained was dissolved in chloroform, 0.5 ml of trifluoroaceticacid was added, and the solid which separated was rinsed withchloroform. This was re-precipitated from chloroform/methanol=1/1solution to give 0.2 g of1,12-(dodecanedioxy)bis(3′-phosphatidyl-2′-deoxythymidine) (Compound 1)as a white powder (yield 35%).

The ¹H-NMR spectrum of this compound is shown in FIG. 1. In the ¹H-NMR(heavy water, 25° C.), signals due to the methylene groups of long chainalkyl groups having a σ value of 1.2–1.3 ppm, 1.6 ppm, 3.8 ppm, andsignals due to the methyl protons bonded at the 5 position of thepyrimidine base in the vicinity of 1.9 ppm, the protons bonded at the 2′position of deoxyribose in the vicinity of 2.4 and 2.6 ppm, themethylene protons bonded to the 5′ position of deoxyribose in thevicinity of 3.8 ppm, the proton bonded to the 4′ position of deoxyribosein the vicinity of 4.2 ppm, the proton bonded to the 3′ position ofdeoxyribose in the vicinity of 4.8 ppm, the proton bonded to the 1′position of deoxyribose in the vicinity of 6.3 ppm, the proton bonded tothe 6 position of the pyrimidine base in the vicinity of 7.7 ppm, andthe imido group NH proton bonded to the 3 position of the pyrimidinebase in the vicinity of 9.2 ppm, were respectively observed.

The physical properties and detailed mass spectral analysis results forthis compound are as follows.

Rf value of thin layer chromatography=0.71(chloroform/methanol=1/1)

Melting point=232° C. (decomposition)

Detailed mass spectrum analysis value (as [M−H+]-), calculated value:809.2775, experimental value: 809.2770.

Manufacturing Example 2

An identical procedure to that of Manufacturing Example 1 was performedusing 1,11-undecane carboxylic acid instead of 1,10-decane dicarboxylicacid, and 1,13-(tridecanedioxy)bis-(3′-phosphatidyl-2′-deoxythymidine)(Compound 2) was obtained (yield 40%).

The physical properties and detailed mass spectral analysis results forthis compound are as follows.

Rf value of thin layer chromatography=0.70(chloroform/methanol=1/1)

Melting point=225° C. (decomposition)

Detailed mass spectrum analysis value (as [M−H+]-), calculated value:823.2932, experimental value: 823.2937.

Manufacturing Example 3

An identical procedure to that of Manufacturing Example 1 was performedusing 1,12-dodecane dicarboxylic acid instead of 1,10-decanedicarboxylic acid, and1,14-(tetradecanedioxy)bis-(3′-phosphatidyl-2′-deoxythymidine) (Compound3) was obtained (yield 25%).

The physical properties and detailed mass spectral analysis results forthis compound are as follows.

Melting point=230° C. (decomposition)

Rf value of thin layer chromatography=0.70(chloroform/methanol=1/1)

Detailed mass spectrum analysis value (as [M−H+]-), calculated value:837.3088, experimental value: 837.3074.

Manufacturing Example 4

An identical procedure to that of Manufacturing Example 1 was performedusing 1,13-tridecane dicarboxylic acid instead of 1,10-decanedicarboxylic acid, and1,15-(pentadecanedioxy)-bis(3′-phosphatidyl-2′-deoxythymidine) (Compound4) was obtained (yield 36%).

The physical properties and detailed mass spectral analysis results forthis compound are as follows.

Rf value of thin layer chromatography=0.70(chloroform/methanol=1/1)

Melting point=232° C. (decomposition)

Detailed mass spectrum analysis value (as [M−H+]-), calculated value:851.3245, experimental value: 851.3255.

Manufacturing Example 5

An identical procedure to that of Manufacturing Example 1 was performedusing 1,14-tetradecane dicarboxylic acid instead of 1,10-decanedicarboxylic acid, and1,16-(hexadecanedioxy)-bis(3′-phosphatidyl-2′-deoxythymidine) (Compound5) was obtained (yield 30%).

The physical properties and detailed mass spectral analysis results ofthis compound are as follows.

Melting point=227° C. (decomposition)

Rf value of thin layer chromatography=0.67(chloroform/methanol=1/1)

Detailed mass spectrum analysis value (as [M−H+]-), calculated value:865.3401, experimental value: 865.3423.

Manufacturing Example 6

An identical procedure to that of Manufacturing Example 1 was performedusing 1,16-hexadecane dicarboxylic acid instead of 1,10-decanedicarboxylic acid, and1,18-(octadecanedioxy)bis-(3′-phosphatidyl-2′-deoxythymidine) (Compound6) was obtained (yield 20%).

The physical properties and detailed mass spectral analysis results ofthis compound are as follows.

Melting point=230° C. (decomposition)

Rf value of thin layer chromatography=0.62(chloroform/methanol=1/1)

Detailed mass spectrum analysis value (as [M−H+]-), calculated value:893.3714, experimental value: 893.3719.

Manufacturing Example 7

An identical procedure to that of Manufacturing Example 1 was performedusing 1,18-octadecane dicarboxylic acid instead of 1,10-decanedicarboxylic acid, and1,20-(icosanedioxy)bis-(3′-phosphatidyl-2′-deoxythymidine) (Compound 7)was obtained (yield 36%).

The physical properties and detailed mass spectral analysis results ofthis compound are as follows.

Melting point=233° C. (decomposition)

Rf value of thin layer chromatography=0.60(chloroform/methanol=1/1)

Detailed mass spectrum analysis value (as [M−H+]-), calculated value:921.4027, experimental value: 921.4022.

Example 1

100 mg of Compound 1(1,12-(dodecanedioxy)bis(3′-phosphatidyl-2′-deoxythymidine) obtained inManufacturing Example 1 was introduced into a sample bottle containing0.5 ml of water, and dissolved while maintaining the temperature at 60°C. or more. After leaving the resulting aqueous solution at roomtemperature, the aqueous solution solidified after several days and thedesired hydrogel was thus obtained.

Example 2

An identical procedure to that of Example 1 was followed using Compound2 1,13-(tridecanedioxy)bis(3′-phosphatidyl-2′-deoxythymidine) obtainedin Manufacturing Example 2, and a hydrogel was obtained.

Example 3

1 mg of Compound 7(1,20-(icosanedioxy)bis(3′-phosphatidyl-2′-deoxythymidine) obtained inManufacturing Example 7 was introduced into a sample bottle containing0.5 ml of water. While maintaining the temperature at 60° C. or more, itwas irradiated with ultrasound for one hour and the compound wasdissolved. After leaving the resulting aqueous solution at roomtemperature, the aqueous solution solidified after several days and thedesired hydrogel was thus obtained. A scanning electron micrograph ofthe freeze-dried hydrogel is shown in FIG. 2.

Examples 4–10

The gelatinizing ability was examined for aqueous solutions, of varyingpH, of Compounds 1–7 synthesized in Manufacturing Examples 1–7. 10 mg ofthese compounds was placed in a sample bottle together with 0.5 ml ofbuffer solution adjusted to various pH, and while maintaining thetemperature at 60° C. or more, the mixture was irradiated withultrasound for one hour. After leaving the resulting aqueous solution atroom temperature, the formation of a gel was observed. These results areshown in Table 1.

TABLE 1 Compound pH 168 pH 401 pH 755 pH 918 milliQ water 1 I S S S S 2I S S S S 3 I S S S S 4 I S S S P 5 I S S S P 6 I P P S G 7 I LG G G G

In the table, the formation of gel is indicated by “G”, partialformation of gel is indicated by “LG”, unchanged aqueous solution isindicated by “S”, the formation of a precipitate is indicated by “P”,and insolubility is indicated by “I”.

It is seen that when the concentration of hydrogelatinizer is 2wt %,Compound 6 and Compound 7 efficiently solidified milliQ water (distilledwater) or water of pH 4–9, and the desired hydrogel was obtained.

1. A hydrogelatinizer represented by the general formula:R-A_(n) wherein each A is a nucleotide, which may be identical to ordifferent from the other A group(s), n is 2 or 3, and R is a hydrocarbonchain having 12–20 carbon atoms that is bivalent when n is 2 andtrivalent when n is 3, said hydrocarbon being bonded to a phosphoricacid part of said nucleotides.
 2. A hydrogelatinizer represented by thegeneral formula:B—R—C where, B and C, which may be identical or different, arerespectively nucleotides and R is a bivalent hydrocarbon chain having12–20 carbon atoms, said hydrocarbon being bonded to a phosphoric acidpart of said nucleotides.
 3. The hydrogelatinizer according to claim 1,wherein said nucleotide is 2′-deoxythymidine-3′-monophosphoric acid. 4.A method of manufacturing the hydrogelatinizer according to claim 1,which comprises the steps of reacting a nucleotide, comprising a sugarpart protected by an acetyl group, 5′-O-4,4′,4″-tris(4-benzoyloxy)tritylgroup or dimethoxytrityl group, with a diol or triol to produce aphosphite ester, oxidizing this phosphite ester with iodine or t-butylhydroperoxide to produce a phosphate ester, and removing the protectivegroup by using an acid.
 5. A hydrogelatinizer according to claim 2,wherein said nucleotide contains only one phosphoric acid group.
 6. Ahydrogelatinizer according to claim 2, wherein said nucleotide is2′-deoxythymidine-3′-monophosphoric acid.
 7. A hydrogelatinizeraccording to claim 5, wherein said nucleotide is2′-deoxythymidine-3′-monophosphoric acid.
 8. A method of manufacturingthe hydrogelatinizer according to claim 2, which comprises the steps ofreacting a nucleotide, comprising a sugar part protected by an acetylgroup, 5′-O-4,4′,4″-tris(4-benzoyloxy)trityl group or dimethoxytritylgroup, with a diol or triol to produce a phosphite ester, oxidizing thisphosphite ester with iodine or t-butyl hydroperoxide to produce aphosphate ester, and removing the protective group by using an acid. 9.A method according to claim 4, wherein said nucleotide contains only onephosphoric acid group.
 10. A method of according to claim 9, whereinsaid nucleotide is 2′-deoxythymidine-3′-monophosphoric acid.
 11. Amethod according to claim 8, wherein said nucleotide contains only onephosphoric acid group.
 12. A method of claim 11, wherein said nucleotideis 2′-deoxythymidine-3′-monophosphoric acid.
 13. A method ofmanufacturing the hydrogelatinizer according to claim 1, which comprisesthe steps of reacting a nucleotide comprising a sugar part having aprotective group and a phosphoric acid part, with a diol or triol toproduce a phosphite ester, oxidizing this phosphite ester to produce aphosphate ester, and removing the protective group from the sugar partof the nucleotide.
 14. The method of claim 13 wherein the phosphoricacid part of the nucleotide has a protective group.
 15. The method ofclaim 14 wherein the phosphoric acid part of the nucleotide is protectedwith an amidite group.
 16. A method of manufacturing thehydrogelatinizer according to claim 2, which comprises the steps ofreacting a nucleotide comprising a sugar part having a protective groupand a phosphoric acid part, with a diol or triol to produce a phosphiteester, oxidizing this phosphite ester to produce a phosphate ester, andremoving the protective group from the sugar part of the nucleotide. 17.The method of claim 16 wherein the phosphoric acid part of thenucleotide has a protective group.
 18. The method of claim 17 whereinthe phosphoric acid part of the nucleotide is protected with an amiditegroup.
 19. A hydrogel comprising water and an effective amount of thehydrogelatinizer of claim
 1. 20. A hydrogel comprising water and aneffective amount of the hydrogelatinizer of claim 2.